In Article <9309020012.AA27496 at net.bio.net>
AMCARTHU at UVVM.UVIC.CA (Andrew McArthur) writes:
>Hello,
>>I am working on sequencing a 600 b.p. PCR product by using asymetric PCR
>to make ssDNA and then using Sequenase (U.S.B.) to sequence. I am getting
I have tried using the asymetric PCR single-stranded approach to getting
Sequencable template, but get better results with
heat denaturation and primer annealing on dry ice (Casanova et al published a
paper on this in Nucleic Acids Research in 1989). This approach is, i think,
nicer for a couple of reasons: you do one PCR, and therefore lessen the chance
of PCR artifacts
Try this, After the first PCR, run the product in 2% LMP
agarose and cut the band out. Put it thru a Promega magic prep column,
following the promega protocol (These things are cheap and give good
recovery and
real clean DNA). Run it on a gel again against a marker of known conc (I use
PhiX174) and calculate the conc, then work out a 25-50:1 picomolar ratio
Primer:PCR product, in 8uL. Boil the mix for about 5min (or 95C in PCR
machine) and snap cool it on
dry ice (30 secs) to let the primer beat the other strand to anneal to the
target strand. Then put the annealed mix onwet ice and carry on the sequenase
protocol.
I find this works better than asymetric PCR, and cleaning up dsDNA is easier
than ssDNA (as far as getting good templates for sequencing goes anyway).
Another thing you may want to try is to generate ssDNA by using lambda
exonuclease to chew away one strand with a phosphorylated primer. A good paper
on this appeared in 1993 Biotechniques (sorry, don't have full refn handy).
Brian Scrimshaw
Wellington
New Zealand